The present invention relates to a cell culturing support and a process for fabricating the same.
At present, cell culture of various kinds of animals and plants is prevalent, and novel cell culture methods are being developed. The cell culture technique is utilized, for example, for elucidating the biochemical phenomena and nature of cells or for manufacturing useful substances. Further, attempts of investigating physiological activities or toxicity of the agents artificially synthesized using cultured cells.
Some of the cells, particularly most of the animal cells, have adhesion dependency in that they grow adhering onto something, and they cannot be kept alive for a long time in a floating state in vitro. Culturing of such cells having adhesion dependency requires a carrier on which the cells are to be adhered, and plastic Petri dishes coated uniformly with an adhesive protein such as collagen and fibronectin are generally employed for such purpose.
It is known that these adhesive proteins act Upon the culturing cells to influence the cell adhesion rate and cell adhesion form of the cells.
Further, various kinds of substances which specifically act on some sorts of cells have been found quite recently. For example, an artificial substrate material poly-N-vinyl-benzyl-D-lactonamide (PVLA) terminated with galactose is synthesized and utilized for hepatocyte culturing ("JINKO ZOKI (Artificial Organs)", vol. 19, No. 3 (1990) pp. 1156-1160). This artificial substrate material binds specifically hepatocytes and serves to maintain their spherical form.
Meanwhile, also reported is a technique in which cells to be cultured are adhered and arranged only onto a very small area of the support. Such technique enables application of the cultured cells to artificial internal organs, biosensors, bioreactors, etc. The most important technique in the patterning of culturing cells is the treatment of the cell culturing support, and some methods are currently tried.
For example, Japanese Unexamined Patent Publication No. 245181/1990 discloses application of an electric charge retaining medium having formed thereon an electrostatic charge pattern to cell culture. Meanwhile, Japanese Unexamined Patent Publication No. 7576/1991 discloses a technique of forming a hydrophilic or hydrophobic pattern on a support, in which a photosensitive hydrophilic polymer having no cell adhesiveness is patterned by photolithography in order to arrange cultured cells thereon as desired.
Meanwhile, Japanese Unexamined Patent Publication No. 7577/1991 discloses a technique of introducing a cell-adhesive functional group to the cell culture support, in which an ultraviolet ray or a radioactive ray is irradiated onto a cell culturing material having a cell-nonadhesive surface so as to introduce a cell-adhesive group thereto, or an ultraviolet ray or a radioactive ray is irradiated onto a cell culturing material to introduce a polymerization initiating species, followed by polymerization of a cell-adhesive or cell-nonadhesive monomer on said species.
The technique of patterning a functional organic film such as an enzyme film on the surface of a support is practically utilized in the field of biosensor manufacturing. For example, Japanese Patent Application No. 209165/1984 discloses patterning of an enzyme film by the lift-off method at the sensitive portion of a semiconductor ionsensor. The lift-off method consists of a step of applying a photoresist onto the surface of a sensor, a step of removing by photolithography the photoresist at a desired positions where an enzyme film is to be formed, a step of forming the enzyme film and a step of dissolving the photoresist.
The conventional cell culturing supports have a pattern formed by utilizing presence and absence .of electrostatic charge or hydrophilicity/hydrophobicity difference. However, such support has only two types of surfaces, i.e. one to which cells adhere well and one to which cells do not substantially adhere, so that if cell culture is carried out using such support, only a simple pattern can be obtained depending on the feasibility of cell adhesion. In other words, no cell culturing support having a highly functional patterned surface, for example, a surface capable of showing various rates of cell adhesion and a surface capable of influencing the cell adhesion form, which is supposed to significantly influence the cell function, has yet been developed.
Meanwhile, the patterned surface formed by utilizing the presence and absence of electrostatic charge or hydrophilicity/hydrophobicity difference, can show a low selectivity depending on the kinds of cells. Accordingly, it is difficult to allow plural kinds of cells to adhere onto different surfaces respectively in the conventional supports.
With respect to fabrication of a cell culturing support, while photolithography using a photosensitive polymer is studied, there is disclosed no successful technique of directly patterning by photolithography a substance which influences the cell adhesion rate or cell adhesion form.